Abstract
Intra-tumor hypoxia is a common feature in many solid cancers. Although transcriptional targets of hypoxia-inducible factors (HIFs) have been well characterized, alternative splicing or processing of pre-mRNA transcripts which occurs during hypoxia and subsequent HIF stabilization is much less understood. Here, we identify many HIF-dependent alternative splicing events after whole transcriptome sequencing in pancreatic cancer cells exposed to hypoxia with and without downregulation of the aryl hydrocarbon receptor nuclear translocator (ARNT), a protein required for HIFs to form a transcriptionally active dimer. We correlate the discovered hypoxia-driven events with available sequencing data from pan-cancer TCGA patient cohorts to select a narrow set of putative biologically relevant splice events for experimental validation. We validate a small set of candidate HIF-dependent alternative splicing events in multiple human gastrointestinal cancer cell lines as well as patient-derived human pancreatic cancer organoids. Lastly, we report the discovery of a HIF-dependent mechanism to produce a hypoxia-dependent, long and coding isoform of the UDP-N-acetylglucosamine transporter SLC35A3.
Highlights
We identify many hypoxiainducible factors (HIFs)-dependent alternative splicing events after whole transcriptome sequencing in pancreatic cancer cells exposed to hypoxia with and without downregulation of the aryl hydrocarbon receptor nuclear translocator (ARNT), a protein required for HIFs to form a transcriptionally active dimer
Using the hypoxia score across all samples in The Cancer Genome Atlas (TCGA) with available RNA-Seq measurements, we associated the hypoxia score with the PSI or expression values of the events we identified as HIF-specific in our experiment using the two models displayed below
To assess the contribution of all three HIFα proteins to gene expression and alternative splicing output during hypoxia, we knocked down their dimerization partner ARNT with ARNT-specific siRNA in the human pancreatic cancer cell line AsPC1
Summary
The most important and well-studied proteins involved in oxygen sensing and signaling include hypoxia-inducible factors (HIFs) and their regulators [3]. HIF1α and HIF2α proteins are a class of tran scriptional activators which, under hypoxic conditions, dimerize with ARNT/HIF1β [3,4] and form a transactivation complex with p300/CBP [5] to regulate gene expression of thousands of genes involved in various cellular pathways, including angiogenesis [6,7], proliferation [7,8,9], metabolism [10,11,12], apoptosis [13,14] and DNA repair [15]. It has been repeatedly shown that cancer cells, utilizing HIF transcriptional programs, are able to gain competitive advantages over normal cells through metabolic adapta tion, growth optimization, immune system evasion, and better survival in pathophysiological microenvironments [20,21,22,23,24,25]
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